Cellular gels. Purifying and mapping long DNA molecules

Abstract

Long DNA molecules of greater than 10(5) bp (0.1 Mbp) are easily broken by pipetting. Therefore, chromosomal DNA is generally isolated after embedding cells in a protective coat of agarose. The embedded DNA can then be cut into long pieces and fractionated on gels using pulsed fields, but these pieces are again easily broken if the resolved DNA molecules are recovered from the gels. We now describe a novel gel matrix, a 'cellular' gel, that permits the recovery of resolved fragments from gels in a form that enables facile manipulation without shear. This facilitates purification and restriction mapping of fragments of 0.1-1.0 Mbp. We illustrate the utility of the method by mapping chromosome III of baker's yeast, which has a length of approximately 0.36 Mbp. This method should facilitate purification and restriction mapping of yeast artificial chromosomes.

HAPPY mapping of a YAC reveals alternative haplotypes in the human immunoglobulin VHlocus

“…Yeast cells (Saccharomces cerevisiae, strain AB1380) containing the YAC IGH2 were grown at 30°C to mid-logarithmic phase in rich medium lacking uracil, spheroplasted and encapsulated in agarose microbeads at a final density of 106cells/ml of agarose (15). Microbeads were washed 6 times at intervals of 30 minutes in lOmM Tris (pH8.0), 1mM Na2EDTA, 1% w/v lithium dodecyl sulphate to remove cellular proteins, and stored at 4°C in TE (1OmM Tris.HCl pH 8.0, 1mM Na2EDTA).…”

Nanodroplet real-time PCR system with laser assisted heating

“…However, there are 300–400 copies of the genes encoding 18S ribosomal RNA occurring in five pairs of chromosomes in each human genome [26, 27]. Furthermore, pipetting can readily produce hydrodynamic shearing of DNA longer than approximately 100,000 base pairs [28], facilitating distribution of the 18S gene during dilution. Thus, the 40 pg/μl droplets still contain 120–160 copies of target templates, and it is not surprising that all the droplets containing only 0.4 copies of human genome were successfully amplified.…”